Fauver et al. teaches an optical imaging system that generates pseudoprojection images from multiple views of a specimen, such as a biological cell. Careful preparation of samples is required prior to initiating optical imaging. To generate a pseudoprojection for each view, the microscope objective lens is scanned in a linear direction that is orthogonal to the central axis of the capillary tube or cylindrical sample volume. To generate multiple pseudoprojections at different views, the cylindrical sample is rotated about its central axis and the procedure for taking a pseudoprojection is repeated. The cylindrical sample used is a line of single cells that are held in a refractive-index-matching gel within a microcapillary tube. In other embodiments, such as disclosed in Fauver et al., the microscope objective is scanned by moving it in a range approximating the tube's inner diameter, at a frequency of less than 100 Hz, using commercially available single axis scanners. A method for substantially increased specimen scanning would be advantageous since scanning an objective lens is a significant rate-limiting step for imaging cells using pseudoprojection technology.
Since standard optical microscope objective lenses are much larger and heavier than a typical cylindrical sample that may be composed of, for example, cells embedded within a rigid polymer thread, one purpose of the present invention is to scan the sample and not the significantly more massive objective lens. Optical fibers of the same material, such as fused silica and size, for example, on the order of 125 microns diameter, can be moved at resonance in the 10 kHz frequency range. As a result, single axis scanning speed may be increased up to 100 times over the previous embodiments using the techniques of the present invention.
In 3D imaging for optical tomography there is a need for improving the image quality of a projection image acquired from a thick sample. There are a number of detrimental optical aberrations that degrade images including:    (a) lateral smearing of the image due to inherent optical system limitations which occurs even for thin specimens;    (b) lateral blurring of the image due to the contributions from the out-of-focus portions of the specimen which is not an issue for thin specimens;    (c) artifactual smearing of the image in the plane perpendicular to the axis of rotation due to using a tomographic backprojection reconstruction algorithm; and    (d) loss of sensitivity when some regions of the volume contain no features.
Lateral smearing of the image, also known as diffraction degradation, is typically present in optical systems. It is described by the system modulation transfer function (MTF). Lateral smearing is a function of, among other things, diffraction through the system's aperture, lens aberrations, and detector pixel size. Typically, it is corrected by measuring a two-dimensional point-spread function (2D PSF) for the system, and deconvolving the image with the 2D PSF electronically in post-acquisition processing.
Lateral blurring, also referred to herein as defocus blurring, is typically present for any sample with a finite thickness. As with lateral smearing, lateral blurring is usually corrected in post-acquisition processing, if at all.
Artifactual smearing is a result of using a backprojection technique in computing a 3D reconstruction of the image as taught, for example, in Fauver et al. A backprojection algorithm typically operates to spread the measured image along the optical axis. If backprojection is done from a large number of observation angles, the PSF becomes artificially smeared.
Loss of sensitivity, or biasing, is unique to optical tomography, as it results from focusing in planes that contain no features. Focusing on such regions may be unavoidable as where the location of an object of interest cannot be readily determined a priori. One such example includes a case where a cell has a diameter of about ten microns, residing within a much larger microcapillary tube, typically having a diameter of about 40 microns. The result is an increase in the DC or zero-spatial frequency component in the image's power spectrum, unaccompanied by any increase of the non-zero spatial frequencies. For a camera with a limited dynamic range (i.e., limited bits of resolution), features become less distinguishable as more and more of them share the same gray-level in an acquired image.
Lateral smearing, lateral blurring, and artifactual smearing of the image can be corrected using post processing software, albeit with some loss of precision when the data acquired is digitized to a finite number of bits. However, loss of sensitivity is more difficult to correct in software, because once the measurement precision is lost, it cannot be readily restored without a priori knowledge. The present invention provides for the first time a method for analog pre-processing of projection images wherein a spatial filter mask, having appropriate optical densities distributed over its surface, is placed in one or more Fourier planes (i.e., aperture stops) of an optical system.